Ultracentrifugal Fractionation Research Articles

Cellulase mediated stress triggers the mutations of oleaginous yeast Trichosporon cutaneum with super-large spindle morphology and high lipid accumulation.

Accumulation of intracellular lipid bodies in oleaginous yeast cells is highly restricted by their natural intracellular space. Here we show a cellulase mediated adaptive evolution with ultra-centrifugation fractionation of oleaginous yeast Trichosporon cutaneum to obtain the favorable cell structure for lipid accumulation. Cellulase was added to the wheat straw hydrolysate during long-term adaptive evolution for disruption of cell wall integrity of T. cutaneum cells. The cellulase, together with ultracentrifugation force, triggered multiple mutations and transcriptional expression changes of the functional genes associated with cell wall integrity and lipid synthesis metabolism. The fractionated mutant T. cutaneum YY52 demonstrated the heavily weakened cell wall and high lipid accumulation by the super-large expanded spindle cells (two orders of magnitude greater than the parental). A record-high lipid production by T. cutaneum YY52 was achieved (55.4±0.5gL-1 from wheat straw and 58.4±0.1gL-1 from corn stover). This study not only obtained an oleaginous yeast strain with industrial application potential for lipid production but also provided a new method for generation of mutant cells with high intracellular metabolite accumulation.

Comprehensive isolation of extracellular vesicles and nanoparticles.

There is an increasing appreciation for the heterogeneous nature of extracellular vesicles (EVs). In addition, two nonvesicular extracellular nanoparticles (NVEPs), exomeres and supermeres, have been discovered recently that are enriched in many cargo previously ascribed to EVs. The EV field has largely focused on EV isolation and characterization, while studies on NVEPs are limited. At this juncture, it is critically important to have robust and reliable methods to separate distinct populations of EVs and NVEPs to assign cargo to their correct carrier. Here, we provide a comprehensive step-by-step protocol for sequential isolation of large and small EVs, nonvesicular fractions, exomeres and supermeres from the same starting material. We describe in detail the use of differential ultracentrifugation, filtration, concentration and high-resolution density-gradient fractionation to obtain purified fractions of distinct populations of EVs and NVEPs. This protocol allows assignment and enrichment of a biomolecule of interest to its specific extracellular compartment. Compared to other isolation methods, our protocol has unique advantages, including high purity and reproducibility, with minimal expertise required. The protocol can be applied to purification of EVs and NVEPs from cell culture medium and human plasma and requires ~72 h to complete. Adoption of this protocol will help translational investigators identify potential circulating biomarkers and therapeutic targets for a host of human diseases and allow basic scientists to better understand EV and NVEP biogenesis and function. Overall, this protocol will allow those interested in isolating EVs and extracellular particles to advance scientific inquiry to answer outstanding questions in the field.

Uptake of oomycete RXLR effectors into host cells by clathrin-mediated endocytosis.

Filamentous (oomycete and fungal) plant pathogens deliver cytoplasmic effector proteins into host cells to facilitate disease. How RXLR effectors from the potato late blight pathogen Phytophthora infestans enter host cells is unknown. One possible route involves clathrin-mediated endocytosis (CME). Transient silencing of NbCHC, encoding clathrin heavy chain, or the endosome marker gene NbAra6 encoding a Rab GTPase in the model host Nicotiana benthamiana, attenuated P. infestans infection and reduced translocation of RXLR effector fusions from transgenic pathogen strains into host cells. By contrast, silencing PP1c isoforms, susceptibility factors not required for endocytosis, reduced infection but did not attenuate RXLR effector uptake. Endosome enrichment by ultracentrifugation and sucrose gradient fractionation revealed co-localization of RXLR effector Pi04314-RFP with clathrin-coated vesicles. Immunopurification of clathrin- and NbAra6-associated vesicles during infection showed that RXLR effectors Pi04314-RFP and AvrBlb1-RFP, but not apoplastic effector PiSCR74-RFP, were co-immunoprecipitated during infection with pathogen strains secreting these effectors. Tandem mass spectrometry analyses of proteins co-immunoprecipitated with NbAra6-GFP during infection revealed enrichment of host proteins associated with endocytic vesicles alongside multiple pathogen RXLR effectors, but not apoplastic effectors, including PiSCR74, which do not enter host cells. Our data show that the uptake of P. infestans RXLR effectors into plant cells occurs via CME.

Equine osteoarthritis modifies fatty acid signatures in synovial fluid and its extracellular vesicles

BackgroundIndividual fatty acids (FAs) and their derivatives (lipid mediators) with pro-inflammatory or dual anti-inflammatory and pro-resolving properties have potential to influence the health of joint tissues. Osteoarthritis (OA) is an age-associated chronic joint disease that can be featured with altered FA composition in the synovial fluid (SF) of human patients. The counts and cargo of extracellular vesicles (EVs), membrane-bound particles released by synovial joint cells and transporting bioactive lipids, can also be modified by OA. The detailed FA signatures of SF and its EVs have remained unexplored in the horse — a well-recognized veterinary model for OA research.MethodsThe aim of the present study was to compare the FA profiles in equine SF and its ultracentrifuged EV fraction between control, contralateral, and OA metacarpophalangeal joints (n = 8/group). The FA profiles of total lipids were determined by gas chromatography and the data compared with univariate and multivariate analyses.ResultsThe data revealed distinct FA profiles in SF and its EV-enriched pellet that were modified by naturally occurring equine OA. Regarding SFs, linoleic acid (generalized linear model, p = 0.0006), myristic acid (p = 0.003), palmitoleic acid (p < 0.0005), and n-3/n-6 polyunsaturated FA ratio (p < 0.0005) were among the important variables that separated OA from control samples. In EV-enriched pellets, saturated FAs palmitic acid (p = 0.020), stearic acid (p = 0.002), and behenic acid (p = 0.003) indicated OA. The observed FA modifications are potentially detrimental and could contribute to inflammatory processes and cartilage degradation in OA.ConclusionsEquine OA joints can be distinguished from normal joints based on their FA signatures in SF and its EV-enriched pellet. Clarifying the roles of SF and EV FA compositions in the pathogenesis of OA and their potential as joint disease biomarkers and therapeutic targets warrants future studies.

A facile strategy for comprehensive proteome analysis of urine using ultracentrifugation fractionation, solid-phase alkylation based sample preparation and liquid chromatography-mass spectrometry

In this work, a facile strategy with combination of ultracentrifugation fraction, solid-phase alkylation sample preparation and liquid chromatography-mass spectrometry (LC-MS) was developed to achieve comprehensive proteome profiling of urine (CPU). By this strategy, a total of 1659 proteins could be identified using a short LC gradient of ∼1h, 2.3 times more than those (730) obtained from raw urine without fractionation. Compared to the current existing methods for urine sample preparation, not only the analysis time could be greatly shortened by 3–4 times, but also the identification coverage of urine proteome was increased by 130%–160%. Such a method was further combined with label-free quantification for the comparative proteome analysis of urine from IgA nephropathy (IgAN) patients and healthy donors, 227 differentially expressed proteins were identified. In addition to 90 proteins that were reported to be closely related to IgAN, we found that multiple members of solute carrier family 22 (SLC22) were up-regulated in IgAN, which might be related to the disturbance of renal metabolic function in patients. All these results demonstrated that our developed method would provide a promising tool for discovering disease related biomarkers in urine.

DodecaRNAs (doRNAs) are abundant in cow’s milk and differentially enriched in milk ultracentrifugation fractions

Background: Extracellular RNAs (exRNAs) are found in numerous extracellular fluids, including milk. Until recently, microRNAs were the focus of research in this area, leaving aside other exRNAs. Recently, a modified small RNA-sequencing (sRNA-seq) approach led to the discovery of very short, 12 and 13 nucleotides (nt) ribosomal RNA (rRNA) fragments (rRFs), designated as dodecaRNAs (doRNAs), in reference to the number of core nucleotides (12 nt) they contain. Since milk is highly enriched in extracellular vesicles (EVs) and exRNAs, we inquired about the existence of doRNAs and other very short exRNAs in milk and milk EV (mEV)-enriched ultracentrifugation fractions.

Ultra-centrifugation force in adaptive evolution changes the cell structure of oleaginous yeast Trichosporon cutaneum into a favorable space for lipid accumulation.

Microbial lipid production from lignocellulose biomass provides an essential option for sustainable and carbon-neutral supply of future aviation fuels, biodiesel, as well as various food and nutrition products. Oleaginous yeast is the major microbial cell factory but its lipid-producing performance is far below the requirements of industrial application. Here we show an ultra-centrifugation fractionation in adaptive evolution (UCF) of Trichosporon cutaneum based on the minor cell density difference. The lightest cells with the maximum intracellular lipid content were isolated by ultra-centrifugation fractionation in the long-term adaptive evolution. Significant changes occurred in the cell morphology with a fragile cell wall wrapping and enlarged intracellular space (two orders of magnitude increase in cell size). Complete and coordinate assimilations of all nonglucose sugars derived from lignocellulose were triggered and fluxed into lipid synthesis. Genome mutations and significant transcriptional regulations of the genes responsible for cell structure were identified and experimentally confirmed. The obtained T. cutaneum MP11 cells achieved a high lipid production of wheat straw, approximately five-fold greater than that of the parental cells. The study provided an effective method for screening the high lipid-containing oleaginous yeast cells as well as the intracellular products accumulating cells in general.

Exploring Subcellular Cerebellar Fractions with the Electron Microscope.

Differential ultracentrifugation and subcellular fractionation historically helped to study the components of the cell, to discover new cellular organelles, and to decipher their morphological and molecular properties. In neuroscience, the technique has yielded important results on neuron biochemistry and the mechanisms of synaptic transmission. This Cerebellar Classic is devoted to the pioneering work of Manuel del Cerro, Ray S. Snider, and Mary Lou Oster-Granite, who isolated purified fractions after successive centrifugations of the rat cerebellum from birth to adulthood and studied them under the electron microscope.

Nanoalgosomes: Introducing extracellular vesicles produced by microalgae.

Cellular, inter‐organismal and cross kingdom communication via extracellular vesicles (EVs) is intensively studied in basic science with high expectation for a large variety of bio‐technological applications. EVs intrinsically possess many attributes of a drug delivery vehicle. Beyond the implications for basic cell biology, academic and industrial interests in EVs have increased in the last few years. Microalgae constitute sustainable and renewable sources of bioactive compounds with a range of sectoral applications, including the formulation of health supplements, cosmetic products and food ingredients. Here we describe a newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes. We isolated these extracellular nano‐objects from cultures of microalgal strains, including the marine photosynthetic chlorophyte Tetraselmis chuii, using differential ultracentrifugation or tangential flow fractionation and focusing on the nanosized small EVs (sEVs). We explore different biochemical and physical properties and we show that nanoalgosomes are efficiently taken up by mammalian cell lines, confirming the cross kingdom communication potential of EVs. This is the first detailed description of such membranous nanovesicles from microalgae. With respect to EVs isolated from other organisms, nanoalgosomes present several advantages in that microalgae are a renewable and sustainable natural source, which could easily be scalable in terms of nanoalgosome production.

Distribution of calcium, phosphorus, sulfur, magnesium, potassium, and sodium in major fractions of donkey milk

The aim of this study was to evaluate the concentrations of Ca, P, S, Mg, K, and Na, and their distribution in major fractions of donkey milk (i.e., fat, casein, whey proteins, and aqueous phase). Individual milk samples were collected by mechanical milking from 16 clinically healthy lactating donkeys. Milk yield per milking was recorded and milk gross composition, casein content, and pH were determined. Whole milk samples were centrifuged to separate fat and to obtain skim milk. Skim milk samples were ultracentrifuged to separate a sedimentable casein pellet and to obtain a supernatant whey (soluble) fraction, which was then ultrafiltered to obtain the aqueous phase of donkey milk. Whole milk and the processed samples were analyzed for the aforementioned elements by inductively coupled plasma-mass spectrometry. The concentration of elements associated with fat, casein, and whey proteins was then calculated. All the Na was present in the aqueous phase. The fat fraction in donkey milk carried very little or none of the investigated elements. The majority of Ca (62.9%) and P (53.1%) was associated with casein, and the rest of these elements was mostly present in the aqueous phase. The majority of Mg was present in the aqueous phase, but a relevant part (32.6%) was associated with the casein fraction. No K was associated with casein. On a molar basis, the ratio of colloidal Ca and P to casein (mmol/g of casein) was more than double the values reported in literature for cow milk. The correlation coefficient was negative between milk pH and P in the ultracentrifuged (r = -0.81) and ultrafiltered (aqueous) fraction (r = -0.66). Milk pH correlated positively with colloidal Ca (r = 0.59) and with the ratio of colloidal Ca to casein (mmol/g of casein; r = 0.68). Colloidal Ca and P were positively correlated (r = 0.64). These data suggest that the high ratio of colloidal Ca and P to donkey casein micelles is due to a larger amount of colloidal calcium phosphate bound to casein micelles compared with literature data on cow milk. The percentage of elements associated with whey proteins was less than 5% for Ca, P, and K, but Mg reached approximately 9% of total Mg. The majority of S (63.6%) was associated with whey proteins, and only one-fourth of this element was associated with casein, indicating a higher content of sulfur-containing amino acids in donkey whey proteins than in casein.

Characterization of Coxsackievirus B4 virus-like particles VLP produced by the recombinant baculovirus-insect cell system expressing the major capsid protein.

Coxsackievirus B4 (CV-B4) is suspected to be an environmental factor that has the intrinsic capacity to damage the pancreatic beta cells and therefore causes insulitis and type 1 diabetes (T1D). Although vaccination against CV-B4 could reduce the incidence of this chronic auto-immune disease, there is currently no therapeutic reagent or vaccine in clinical use. By the employment of the Bac-to-Bac® vector system to express the major viral capsid protein, we contributed towards the development of a CV-B4 vaccine by producing CV-B4 virus-like particles (VLPs) from recombinant baculovirus in infected insect cells. In fact Western blot and Immunofluorescence analysis detected the viral protein 1 (VP1) in the cells resulting from the construction of a recombinant bacmid DNA carrying the key immunogenic protein then transfected in the insect cells. Sucrose gradient ultracentrifugation fractions of the infected cell lysates contained the recombinant protein and the electron microscopy demonstrated the presence of VLPs in these sucrose fractions. This study clearly shows for the first time the expression of CVB4 VP1 structure protein alone can form VLPs in the baculovirus-infected insect cell keeping conserved both characteristics and morphology.

Identification of metolachlor mineralizing bacteria in aerobic and anaerobic soils using DNA-stable isotope probing.

The influence of soil environmental factors such as aeration on the ecology of microorganisms involved in the mineralization and degradation of the popular soil-applied pre-emergent herbicide, metolachlor is unknown. To address this knowledge gap, we utilized DNA-based stable isotope probing (SIP) where soil microcosms were incubated aerobically or anaerobically and received herbicide treatments with unlabeled metolachlor or 13C-metolachlor. Mineralization of metolachlor was confirmed as noted from the evolution of 14CO2 from 14C-metolachlor-treated microcosms and clearly demonstrated the efficient utilization of the herbicide as a carbon source. Terminal restriction fragment length polymorphisms (T-RFLP) bacterial community profiling performed on soil DNA extracts indicated that fragment 307bp from aerobic soil and 212bp from anaerobic soil were detected only in the herbicide-treated (both unlabeled metolachlor and 13C-metolachlor) soils when compared to the untreated control microcosms. T-RFLP profiles from the ultracentrifugation fractions illustrated that these individual fragments experienced an increase in relative abundance at a higher buoyant density (BD) in the labeled fractions when compared to the unlabeled herbicide amendment fractions. The shift in BD of individual T-RFLP fragments in the density-resolved fractions suggested the incorporation of 13C from labeled herbicide into the bacterial DNA and enabled the identification of organisms responsible for metolachlor uptake from the soil. Subsequent cloning and 16S rRNA gene sequencing of the 13C-enriched fractions implicated the role of organisms closely related to Bacillus spp. in aerobic mineralization and members of Acidobacteria phylum in anaerobic mineralization of metolachlor in soil.

Molecular and structural analysis via hydrodynamic methods: Cationic poly(2-aminoethyl-methacrylate)s

Pharmaceutically relevant cationic linear poly(2-aminoethyl-methacrylate)s were synthesized by reversible addition-fragmentation chain transfer polymerization resulting in a homologous series of the homopolymers in a wide range of molar masses (10,000<M<130,000gmol−1) and relatively narrow dispersities (Ð:1.3±0.4). The polymers were studied in 0.2MNaCl and 0.2MNaOH in water, applying combined analytical approach comprising of complementary hydrodynamic and optical methods such as analytical ultracentrifugation, intrinsic viscosity, translation diffusion, flow birefringence and asymmetric flow field-flow fractionation (AF4) as well as standard characterization technique (SEC). The efforts were focused on the evaluation of solution (solvation), molecular (absolute molar masses), conformational (equilibrium rigidity, diameter of the polymer chain) and optical (anisotropy of optical polarizability of the monomer unit) characteristics of the polymers. Consequently the absolute values of the molar masses were determined by the sedimentation-diffusion analysis and AF4 resulting in well consistent results. The corresponding scaling relationships in 0.2MNaCl water ([η]=0.0031×M0.83; s0=0.0324×M0.39; D0=3,909×M−0.64) show linear trends in whole range of molar masses revealing random coil conformation of the macromolecules. Conformational characteristics were determined in 0.2MNaCl (equilibrium rigidity A=5.3±1.5nm) and 0.2MNaOH (A=1.3±0.1nm) revealing an influence of uncompensated charges on the polymer conformation.

Polyphosphate nanoparticles on the platelet surface trigger contact system activation

AbstractPolyphosphate is an inorganic polymer that can potentiate several interactions in the blood coagulation system. Blood platelets contain polyphosphate, and the secretion of platelet-derived polyphosphate has been associated with increased thrombus formation and activation of coagulation factor XII. However, the small polymer size of secreted platelet polyphosphate limits its capacity to activate factor XII in vitro. Thus, the mechanism by which platelet polyphosphate contributes to thrombus formation remains unclear. Using live-cell imaging, confocal and electron microscopy, we show that activated platelets retain polyphosphate on their cell surface. The apparent polymer size of membrane-associated polyphosphate largely exceeds that of secreted polyphosphate. Ultracentrifugation fractionation experiments revealed that membrane-associated platelet polyphosphate is condensed into insoluble spherical nanoparticles with divalent metal ions. In contrast to soluble polyphosphate, membrane-associated polyphosphate nanoparticles potently activate factor XII. Our findings identify membrane-associated polyphosphate in a nanoparticle state on the surface of activated platelets. We propose that these polyphosphate nanoparticles mechanistically link the procoagulant activity of platelets with the activation of coagulation factor XII.

NanoRelease: Pilot interlaboratory comparison of a weathering protocol applied to resilient and labile polymers with and without embedded carbon nanotubes

A major use of multi-walled carbon nanotubes (MWCNTs) is as functional fillers embedded in a solid matrix, such as plastics or coatings. Weathering and abrasion of the solid matrix during use can lead to environmental releases of the MWCNTs. Here we focus on a protocol to identify and quantify the primary release induced by weathering, and assess reproducibility, transferability, and sensitivity towards different materials and uses. We prepared 132 specimens of two polymer-MWCNT composites containing the same grade of MWCNTs used in earlier OECD hazard assessments but without UV stabilizer. We report on a pilot inter-laboratory comparison (ILC) with four labs (two US and two EU) aging by UV and rain, then shipping for analysis. Two labs (one US and one EU) conducted the release sampling and analysis by Transmission Electron Microscopy (TEM), Inductively Coupled Plasma- Mass Spectrometry (ICP-MS), Ultraviolet–Visible Spectroscopy (UV–Vis), Analytical Ultracentrifugation (AUC), and Asymmetric Flow Field Flow Fractionation (AF4). We compare results between aging labs, between analysis labs and between materials. Surprisingly, we found quantitative agreement between analysis labs for TEM, ICP-MS, UV–Vis; low variation between aging labs by all methods; and consistent rankings of release between TEM, ICP-MS, UV–Vis, AUC. Significant disagreement was related primarily to differences in aging, but even these cases remained within a factor of two.

Assessment of six commercial plasma small RNA isolation kits using qRT-PCR and electrophoretic separation: higher recovery of microRNA following ultracentrifugation.

Growing interest in blood-borne microRNAs (miRNAs) as biomarkers has led to the introduction of a number of commercial kits for isolating small RNAs from plasma/serum. We sought to compare the efficacy of six such kits in isolating miRNAs from either whole plasma or a plasma-derived ultracentrifugation (UC) fraction from 2 healthy volunteers with some of the results being validated in 10 additional subjects. To assess the overall yield and concentration of isolated small RNAs, we measured the levels of one spiked-in and four endogenous miRNAs by quantitative reverse transcription and polymerase chain reaction (qRT-PCR). We also tested the performance of the Agilent Bioanalyzer small RNA assay with these RNA samples. Additionally, we tested the effects of hemolysis on measured miRNA levels in whole plasma and in the UC fraction. Both the efficiency of RNA isolation and the relative levels of specific miRNAs in different samples varied considerably between the tested extraction methods. Of all kits tested, the QIAGEN miRNeasy kits (Mini and Serum/Plasma kits) and the Macherey-Nagel NucleoSpin kit produced the highest RNA yields. The QIAGEN Exo kit produced lesser yields than what could be extracted from the UC fraction using the QIAGEN miRNeasy kits and the Macherey-Nagel NucleoSpin kit. Bioanalyzer results showed an average correlation of R2 = 0.8 with endogenous miRNA qRT-PCR results, for sample concentrations >40 pg/µl. The levels of the endogenous miRNAs measured in the two volunteer samples were compared with those in a larger group of subjects (n = 10) and found to be typical. Our comparison favors the use of the QIAGEN Serum/Plasma kit and the Macherey-Nagel NucleoSpin kit for plasma miRNA applications. Furthermore, extraction of miRNAs from the UC fraction results in higher yield than extraction from whole plasma.

Improving Blood Plasma Glycoproteome Coverage by Coupling Ultracentrifugation Fractionation to Electrostatic Repulsion-Hydrophilic Interaction Chromatography Enrichment.

Blood plasma is considered to be an excellent source of disease biomarkers because it contains proteins, lipids, metabolites, cell, and cell-derived extracellular vesicles from different cellular origins including diseased tissues. Most secretory and membranous proteins that can be found in plasma are glycoproteins; therefore, the plasma glycoproteome is one of the major subproteomes that is highly enriched with disease biomarkers. As a result, the glycoproteome has attracted much attention in clinical proteomic research. The modification of proteins with glycans regulates a wide range of functions in biology, but profiling plasma glycoproteins on a global scale has been hampered by the presence of low stoichiometry of glycoproteins in a complex high abundance plasma proteome background and lack of effective analytical technique. This study aims to improve plasma glycoproteome coverage using pig plasma as a model sample with a two-step strategy. The first step involves fractionation of the plasma proteins using ultracentrifugation into supernatant and pellet that is believed to contain low abundant glycoproteins. In the second step, further enrichment of glycopeptides was achieved in both fractions by adopting electrostatic repulsion hydrophilic interaction chromatography (ERLIC) coupled to tandem mass spectrometry (LC-MS/MS) analysis. The coverage of enriched glycoproteins in supernatant, pellet, and whole plasma sample as control was compared. Using this simple sample fractionation approach by ultracentrifugation and further ERLIC enrichment technique, sample complexity was reduced and glycoproteome coverage was significantly enhanced in supernatant and pellet fractions (by >50%) compared with whole plasma sample. This study showed that when ultracentrifugation is coupled to ERLIC glycopeptides enrichment and glycoproteome identification are significantly improved. This study demonstrates the combination of ultracentrifugation and ERLIC as a useful method for discovering plasma glycoprotein disease biomarkers.

Elucidating carbon uptake from vinyl chloride using stable isotope probing and Illumina sequencing.

Vinyl chloride (VC), a known human carcinogen, is a common and persistent groundwater pollutant at many chlorinated solvent contaminated sites. The remediation of such sites is challenging because of the lack of knowledge on the microorganisms responsible for in situ VC degradation. To address this, the microorganisms involved in carbon assimilation from VC were investigated in a culture enriched from contaminated site groundwater using stable isotope probing (SIP) and high-throughput sequencing. The mixed culture was added to aerobic media, and these were amended with labeled ((13)C-VC) or unlabeled VC ((12)C-VC). The cultures were sacrificed on days 15, 32, and 45 for DNA extraction. DNA extracts and SIP ultracentrifugation fractions were subject to sequencing as well as quantitative PCR (qPCR) for a functional gene linked to VC-assimilation (etnE). The gene etnE encodes for epoxyalkane coenzyme M transferase, a critical enzyme in the pathway for VC degradation. The relative abundance of phylotypes was compared across ultracentrifugation fractions obtained from the (13)C-VC- and (12)C-VC-amended cultures. Four phylotypes were more abundant in the heavy fractions (those of greater buoyant density) from the (13)C-VC-amended cultures compared to those from the (12)C-VC-amended cultures, including Nocardioides, Brevundimonas, Tissierella, and Rhodoferax. Therefore, both a previously identified VC-assimilating genus (Nocardioides) and novel microorganisms were responsible for carbon uptake. Enrichment of etnE with time was observed in the heavy fractions, and etnE sequences illustrated that VC-assimilators harbor similar Nocardioides-like etnE. This research provides novel data on the microorganisms able to assimilate carbon from VC.

Techniques to improve detection and analysis of extracellular vesicles using flow cytometry.

Extracellular vesicles (EVs) range in size from 50 nm to 1 µm. Flow cytometry (FCM) is the most commonly used method for analyzing EVs; however, accurate characterization of EVs remains challenging due to their small size and lack of discrete positive populations. Here we report the use of optimization techniques that are especially well-suited for analyzing EVs from a high volume of clinical samples. Utilizing a two pronged approach that included 1) pre-filtration of antibodies to remove aggregates, followed by 2) detergent lysis of a replicate sample to account for remaining false positive events, we were able to effectively limit false positive non-EV events. In addition, we show that lysed samples are a useful alternative to isotypes for setting gates to exclude background fluorescence. To reduce background, we developed an approach using filters to "wash" samples post-staining thus providing a faster alternative to ultracentrifugation and sucrose gradient fractionation. In conclusion, use of these optimized techniques enhances the accuracy and efficiency of EV detection using FCM.

Unconventional secretion of synuclein-γ promotes tumor cell invasion.

Synuclein-γ (SNCG) is a chaperone protein and exists mainly in the cytoplasm. SNCG confers chemoresistance, and is a potential unfavorable biomarker for multiple types of cancer. Our previous work demonstrated that SNCG could be detected in the serum of cancer patients and the medium of cultured cancer cells, but the mechanism of SNCG secretion and its biological roles are unknown. Here, we showed that SNCG levels in the culture medium were positively correlated with cancer cell densities and the concentrations of fetal bovine serum added. SNCG secretion was unaffected by brefeldin A, an inhibitor of the classic protein transport pathway, but was antagonized by exosome inhibitor, lysosome inhibitor, ABC transporter inhibitor, and phosphatidylinositide 3-kinase inhibitor, and knockdown of Rab27a. Ultracentrifugation fractionation revealed that intracellular SNCG was present as both free and vesicle-associated forms, but that the extracellular SNCG was mainly free. The results of reciprocal coimmunoprecipitation experiments showed an interaction between SNCG and flotillin-2, a marker of exosomes and lipid rafts. Moreover, we demonstrated that SNCG, both secreted from tumor cells and exogenously added, markedly promoted cancer cell migration and invasion, but had no effect on noncancerous cells. These findings suggest that SNCG is actively secreted by cancer cells via an unconventional secretion pathway and contributes to aggressive phenotypes of cancer cells.